This invention relates to a reagent and method for determining total globulin in biological fluids, and in particular to improvements in the glyoxylic reaction for determination of tryptophan or tryptophan-containing proteins.
Determining the total quantity of globulins in a biological fluid, such as serum or plasma, is important in the diagnosis of certain disease states. It is also important in the commercial production and purification of globulins.
The traditional clinical method of determining total globulin in a biological sample, such as serum, has been initial determination of total protein, for example by the biuret reaction, followed by delicate chemical fractionation to precipitate the globulin component, and followed determination of the remaining albumin. Globulin content is then calculated as the difference between these values. The difficulties of the traditional method may be overcome by determining globulin directly. Because the so-called glyoxylic (or Hopkins-Cole) reaction is specific for tryptophan and certain tryptophan derivatives, and because globulin is the only substantial source of tryptophan in serum and many other biological fluids, the glyoxylic reaction provides a convenient direct globulin determination which does not require pretreatment of the sample.
Hopkins and Cole, Proc. Roy. Soc., 68, 21 (1901) and J. Physiol. 27, 418 (1902) indicated that the purple color which was produced by the "Adamkiewicz protein reaction" using a reagent mixture consisting of sulfuric acid and acetic acid was due to the presence of tryptophan or tryptophan-containing protein, and that the essential component which initiated the "Adamkiewicz reaction" was the glyoxylic acid which originated as an impurity from the employed acetic acid. Based upon this observation, Hopkins and Cole renamed the "Adamkiewicz reaction" the "glyoxylic reaction".
Fearon, Biochem, J., 14, 548 (1920), further studied the glyoxylic reaction. He replaced sulfuric acid (which he found to be unsuitable) with acetic acid saturated with HCl gas; phosphoric acid was said to have been used in a few unspecified cases. His work utilized indole, scatole and carbazole in addition to tryptophan. He concluded that the overall glyoxylic reaction is the result of two distinct chemical reactions:
A. A CONDENSATION REACTION BETWEEN TWO MOLECULES OF TRYPTOPHAN OR 3-SUBSTITUTED TRYPTOPHAN DERIVATIVE AND ONE MOLECULE OF GLYOXYLIC ACID TO FORM A MOLECULE OF CONDENSATION PRODUCT, DESIGNATED AS THE LEUCO BASE, AND A MOLECULE OF WATER, AND
B. AN OXIDATION REACTION IN WHICH THE LEUCO BASE FORMED BY THE INITIAL CONDENSATION REACTION UNDERGOES OXIDATION IN THE PRESENCE OF AIR TO FORM A MOLECULE OF COLORED PRODUCT AND ANOTHER MOLECULE OF WATER IN THE REACTION MIXTURE. Fearon represented these two reactions as follows:
a. CONDENSATION REACTION ##SPC1##
b. OXIDATION REACTION ##SPC2##
It is evident from the above representation that the overall glyoxylic reaction requires two moles of test substance and one mole of glyoxylic acid to form one mole of colored derivative and two moles of water, and that the final color produced by the glyoxylic reaction is dependent upon the extent to which the condensation and oxidation reactions are favored in the reaction mixture.
In accordance with Fearon's formulation of the glyoxylic reaction, the function of sulfuric acid is to initiate the condensation reaction that results in the formation of leuco base. However, sulfuric acid also exerts a charring effect on the reactants and thus interferes with their ability to produce the leuco base. To minimize the charring action of sulfuric acid and to reduce the viscosity of the reaction mixture, glacial acetic acid is generally used as a diluent. This, however, impairs the condensing activity of sulfuric acid as well as decreasing the solubility of the test specimen (particularly protein) in the reaction mixture.
The reaction in which the leuco base undergoes oxidation by molecular oxygen of air in the presence of sulfuric acid (which also acts as an oxidizing agent in a variable manner) to form the purple color pigment, is rather sensitive, selective, and sluggish. For example, if the reaction mixture incorporates a strong oxidizing agent, the desired purple colored pigment undergoes further oxidation to form products of nonspecific colors. On the other hand, if the reaction mixture contains only weak oxidizing agents, the purple color formation is not stimulated. Moreover, water formed during the selective oxidation of leuco base also hydrolyzes the purple color generated by the reaction.
From the foregoing discussion, it seems that the final or net color produced in the glyoxylic reaction mixture depends upon the extent to which various desirable and undesirable reactions are favored by the employed reagents, their concentrations, and the reaction conditions.
Ever since the discovery of the glyoxylic reaction, various methods have been developed from time to time for the determination of free tryptophan or proteins containing tryptophan, such as globulins, in biological fluids. These methods employed sulfuric or perchloric acid (both may cause charring) as the condensing agent and glacial acetic acid as the diluent. They also generally add glyoxylic acid and a "sensitizing agent", which appears to facilitate the oxidation reaction. An amount of glyoxylic acid which provides a measurable reaction color proportional to globulin concentration has been termed a "colorimetric amount" of glyoxylic acid. Likewise, an amount of sensitizing agent which detectably enhances the reaction color without masking it has been termed a "sensitizing amount" of the sensitizing agent. Winkler, Z. Physiol. Chem., 288, 50 (1934) employed copper sulfate as a "sensitizing agent". Copper sulfate was subsequently also used by Goldenberg and Drewes, Clin. Chem., 17, 358 (1971), by Goldenberg, U.S. Pat. No. 3,607,081 (1971), and by Shaw and McFarlane, Can. J. Res., 16, 361 (1938). It has been reported that copper sulfate masks the glyoxylic reaction color and also tends to precipitate in the presence of concentrated sulfuric acid. Saifer and Gerstenfeld, Clin. Chem., 10, 970 (1964) replaced sulfuric acid and copper sulfate with perchloric acid and potassium persulfate but this initiated the formation of nonspecific color in the glyoxylic reaction mixture.
Opienska-Blauth et al, Anal. Biochem., 6, 69 (1963), considered added glyoxylic acid to be labile in contact with the sulfuric acid present in the reaction mixture. To avoid this problem, these investigators did not add glyoxylic acid to their reaction mixture. Instead, they employed separate acetic acid and sulfuric acid reagents and added a small amount of ferric chloride to the acetic acid to generate glyoxylic acid in situ, since trivalent iron is known to catalyze the oxidation of acetic acid to glyoxylic acid in the presence of sulfuric acid. These investigators added a substantial amount of water (about twenty percent) to their reaction mixture.
Although several methods have been described for the estimation of free tryptophan or tryptophan-containing proteins, these methods have failed to produce reproducible results and have lacked sensitivity. This could be due to the considerable variations in the reaction conditions, as well as in the composition and water content of the final assay systems that were used in these methods. Because of the instability of the reagent or reagents employed by these methods, and the dependence of the methods on the precise concentrations of the reactants, prior methods have generally required recalibration for each set of determinations made.